Zamboni D S, Mortara R A, Rabinovitch M
Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, UNIFESP, Rua Botucatu, 862, 6th andar, São Paulo, SP 04023-062, Brazil.
J Microbiol Methods. 2001 Jan;43(3):223-32. doi: 10.1016/s0167-7012(00)00223-2.
Coxiella burnetii, the agent of Q fever in man and of coxiellosis in other species, is an intracellular pathogen not yet grown axenically. Confocal laser fluorescence microscopy and morphometry were used to measure relative C. burnetii phase II loads and their intracellular distribution in aldehyde fixed and DAPI stained Vero cell monolayers. The fluorescence of single horizontal optical sections provided useful information on relative loads of bacteria in cells and vacuoles. The relative density of the bacteria in the vacuoles was inferred from ratios of fluorescence to vacuolar section areas. Relative bacterial loads, bacterial densities and section areas of large vacuoles increased exponentially between days 2 and 4 of the infection of gamma-irradiated host cells, stabilized between days 4 and 6, and decreased thereafter. Estimated minimum doubling times were higher for the overall complement of the intracellular organisms (about 12 h) than for bacteria that were confined to larger vacuoles (about 10 h).
伯纳特柯克斯体是人类Q热和其他物种柯克斯体病的病原体，是一种尚未能在无细胞培养基中生长的细胞内病原体。利用共聚焦激光荧光显微镜和形态测量法来测定醛固定并经4',6-二脒基-2-苯基吲哚（DAPI）染色的Vero细胞单层中伯纳特柯克斯体II相的相对载量及其细胞内分布。单个水平光学切片的荧光提供了关于细胞和液泡中细菌相对载量的有用信息。液泡中细菌的相对密度可根据荧光与液泡切片面积的比率推断得出。在γ射线照射的宿主细胞感染后的第2天至第4天，大液泡中的相对细菌载量、细菌密度和切片面积呈指数增加，在第4天至第6天稳定下来，此后下降。细胞内生物体总体群体的估计最小倍增时间（约12小时）高于局限于较大液泡中的细菌的估计最小倍增时间（约10小时）。
